A composition for improving lung diseases and its application

Abstract

The present invention discloses a composition for improving pulmonary diseases and its application, belonging to the technical field of biomedicine. The composition for improving pulmonary diseases comprises L-carnosine and ergothioneine in a mass ratio of (1-2):(1-3). By nebulizing the composition for improving pulmonary diseases to COPD rats, it is verified that the composition can reduce the levels of inflammatory factors TNF-α, IL-1β and IL-6 in bronchoalveolar lavage fluid, improve pulmonary inflammation, increase the expression of OCTN1 in the lungs of rats, inhibit the apoptosis of rat lung epithelial cells, and thus improve lung injury.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a composition for improving lung diseases and application thereof. Background Art

[0002] Chronic obstructive pulmonary disease (COPD) is a common chronic lung disease, generally including chronic bronchitis and emphysema. Most patients suffer from lung damage due to long-term exposure to toxic particles or gases (such as cigarette smoke, air pollution, etc.). The main symptoms of lung damage are chronic cough, sputum, shortness of breath or difficulty breathing.

[0003] Currently, the commonly used western medicines for COPD mainly include expectorants or bronchodilators, which can relieve symptoms of shortness of breath, cough and sputum. However, this method only treats the symptoms and not the root cause. It can only relieve symptoms and cannot fundamentally improve lung inflammation and inhibit apoptosis of alveolar epithelial cells, thereby repairing lung damage. Therefore, a drug that can effectively treat COPD is needed. Summary of the invention

[0004] In order to overcome the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a composition for improving lung diseases and its application, so as to effectively treat COPD.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The invention discloses a composition for improving lung diseases, comprising L-carnosine and ergothioneine in a mass ratio of (1-2):(1-3).

[0007] Preferably, the mass ratio of L-carnosine to ergothioneine is 1:2.

[0008] Preferably, it also includes pharmaceutical excipients or other compatible drugs.

[0009] The invention also discloses the use of the above-mentioned composition for improving lung diseases in the preparation of medicines for improving lung diseases.

[0010] Preferably, the lung disease is chronic bronchitis, bronchiectasis, emphysema or pulmonary fibrosis.

[0011] Preferably, the dosage form of the drug is powder, tablet, pill, capsule, oral solution, spray, powder mist, aerosol or nasal drops.

[0012] Preferably, the composition for improving lung diseases is a composition for reducing the levels of inflammatory factors TNF-α, IL-1β and IL-6 in bronchoalveolar lavage fluid.

[0013] Preferably, the composition for improving lung diseases is a composition for increasing the expression of OCTN1 in the lungs.

[0014] Preferably, the composition for improving lung diseases is a composition for inhibiting apoptosis of lung epithelial cells.

[0015] Preferably, L-carnosine and ergothioneine synergistically improve lung diseases.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides a composition for improving lung diseases, and ergothioneine and L-carnosine are used together to improve lung damage. The composition can 1. effectively reduce lung inflammation and inhibit apoptosis of lung epithelial cells; 2. synergistic enhancement: L-carnosine reduces the levels of inflammatory cytokines TNF-α, IL-1β, and IL-6 in bronchoalveolar lavage fluid, and inflammatory cytokines are important factors in inducing or regulating apoptosis of lung parenchymal cells. Ergothioneine mainly enters the mitochondria through the transporter protein OCTN1 of lung cells, directly removes free radicals, and protects mitochondria, effectively reducing ROS-induced inflammatory reactions and reducing mitochondrial-mediated apoptosis of lung epithelial cells. The two cooperate with each other, synergistically enhance, inhibit lung inflammation and apoptosis of lung epithelial cells from different pathways, and greatly enhance the ability to repair lung damage; 3. Safe ingredients: L-carnosine in food is mostly derived from chicken, beef, and pork, ergothioneine is derived from mushrooms and ergot fungi, and the human body itself contains L-carnosine (muscle and brain tissues contain very high concentrations of L-carnosine) and ergothioneine, so both are very safe. The composition for improving lung disease was administered to COPD rats by nebulization, and it was further verified that the composition could reduce the levels of inflammatory factors TNF-α, IL-1β and IL-6 in alveolar lavage fluid, improve lung inflammation, increase the expression of OCTN1 in rat lungs, inhibit the apoptosis of rat lung epithelial cells, and thus improve lung damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The graph is a graph of the levels of TNF-α, IL-1β, and IL-6 in the BALF of COPD rats of the present invention; wherein A is the level of TNF-α in the BALF of COPD rats, B is the level of IL-1β in the BALF of COPD rats, and C is the level of IL-6 in the BALF of COPD rats, a represents p<0.01 compared with the Con group; b represents p<0.01 compared with the Mod group; c represents p<0.01 compared with the Car group and the EGT group;

[0019] Figure 2is a graph of the expression level of OCTN1 of the present invention; wherein A is the quantification of the protein expression band by ImageJ analysis software, B is the OCTN1 protein expression band, a represents p<0.01 compared with the Con group; b represents p<0.01 compared with the Mod group; c represents p<0.01 compared with the Car group and the EGT group;

[0020] Figure 3 It is a graph of the apoptosis rate of lung epithelial cells of the present invention; wherein, a represents p<0.01 compared with the Con group; b represents p<0.01 compared with the Mod group; c represents p<0.01 compared with the Car group and the EGT group. DETAILED DESCRIPTION

[0021] It should be noted that the term "L-carnosine" in the specification and claims of the present invention and the above-mentioned drawings, including its aliases "β-alanyl-L-histidine" and "carnosine", etc., should be understood to belong to the scope of protection of the present invention.

[0022] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0023] Main reagents and instruments: Ergothioneine (purity ≥ 98%) was purchased from Shenzhen Redline Biotechnology Co., Ltd.; L-carnosine (purity 99%) and lipopolysaccharide (LPS) were purchased from Sigma; rat TNF-α, IL-1β and IL-6 ELISA kits, TUNEL kits were purchased from Wuhan Boster Company; RIPA lysis buffer, BCA protein quantification kit, and ECL developer were purchased from Shanghai Biyuntian; 4% paraformaldehyde was purchased from Beijing Dingguo; rabbit anti-rat OCTN1 antibody was purchased from Biorbyt; rabbit anti-rat β-actin antibody was purchased from Abcam (Shanghai) Trading Co., Ltd.; goat anti-rabbit secondary antibody was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.; PARI JuniorBOY SX (085G3305) compressed nebulizer was purchased from German Bairui Company; optical microscope Olympus BX51 was purchased from Olympus Corporation.

[0024] Animal and grouping: 36 SPF level SD male healthy rats (provided by the Experimental Animal Center of the Fourth Military Medical University) with a body mass of 200 ± 20g were adaptively raised for 1 week, with a temperature of 22-26°C, a humidity of 40%-50%, and artificial lighting for 12h during the day and night, and free drinking water during the feeding period. SD rats were randomly divided into Con group (control group), Mod group (model group), Car group (L-carnosine group), EGT group (thioneine group), Mix group (L-carnosine: thioneine=(1-2): (1-3), preferably 1:2), 6 in each group.

[0025] Experimental method: On the first and 14th day of modeling, rats in the Mod, Car, EGT and Mix groups were slowly instilled with LPS into the trachea using a sterile syringe. 200 μg / 200 μL, rotate the rats left and right to allow LPS to enter the rat lung tissue, and inject an equal volume of normal saline into the control group; on days 2 to 13 and 15 to 28, place the rats in the Mod group, Car group, EGT group and Mix group in a closed box for fumigation, smoke once a day, 12 cigarettes each time, each time for 30 minutes, to obtain a COPD rat model, and administer the drug once 30 minutes before fumigation, and the Con group is not fumigated; the drug administration method is as follows: L-carnosine, ergothioneine, and a combination (L-carnosine: ergothioneine=1:2) are respectively dissolved in 10 mL of normal saline, and the dosage is 80 mg / kg, and a rat atomizer drug delivery instrument is used to perform atomization inhalation treatment on the Car group, EGT group and Mix group rats for 30 minutes, and the Mod group and the Con group inhale an equal volume of normal saline.

[0026] Detection of inflammatory factors in bronchoalveolar lavage fluid (BALF): The day after the experiment, rats were killed by cervical dislocation, and then the chest was opened. The right main bronchus was punctured, and 5 mL of sterile pre-cooled PBS was injected to lavage the left bronchoalveoli for a total of 3 times. The lavage fluid was centrifuged at 4°C 2500r / min for 15min, and the supernatant was collected and then stored at -80°C. The levels of TNF-α, IL-1β and IL-6 cytokines in BALF were detected by ELISA, and the operation procedures were carried out according to the instructions of the rat TNF-α, IL-1β and IL-6 ELISA kits.

[0027] TUNEL method for detecting cell apoptosis: The rat lung tissue was removed, the left lung tissue was separated and fixed with 4% paraformaldehyde, then embedded in wax blocks, and made into paraffin sections with a thickness of 4 μm. After the sections were dehydrated, cell apoptosis was detected according to the TUNEL kit detection instructions. The sections were sealed and observed under a microscope, and the average of the proportion of TUNEL-positive cells in 5 non-overlapping fields of view was calculated, and the cell apoptosis rate was calculated.

[0028] Detection of relative expression of OCTN1 protein in lung tissue: The right lung tissue of rats was ground and lysed with RIPA lysis buffer to extract total lung tissue protein, and then its concentration was determined using a BCA kit. After SDS-PAGE electrophoresis, membrane transfer to PVDF membrane, and blocking, rabbit anti-rat OCTN1 antibody and rabbit anti-rat β-actin antibody were added, and the cells were incubated overnight at 4°C, washed with TBS water 3 times, 15 min each time, and HRP-labeled secondary antibody (i.e., goat anti-rabbit secondary antibody) was added for further incubation for 1 h. Finally, ECL developer was added to develop the protein bands, and the imaging of each group of protein bands was observed, and the protein bands were analyzed using ImageJ analysis software.

[0029] Results analysis see Figure 1 to Figure 3 :

[0030] Under the stimulation of cigarette smoke or other irritants, macrophages and airway epithelial cells secrete proinflammatory cytokines and chemokines such as TNF-α, IL-1β and IL-6, which induce the activation and recruitment of neutrophils and monocytes and the release of other cytokines, thereby promoting inflammation; activated neutrophils and macrophages can promote the development of emphysema by releasing oxygen free radicals and proteases. Airway inflammatory response is of great significance in the progression of COPD, so reducing lung inflammation can effectively improve lung damage.

[0031] Figure 1 Compared with the Con group, the levels of inflammatory factors TNF-α, IL-1β and IL-6 in BALF of the Mod group were significantly increased (p < 0.01), indicating that the rat COPD model was successfully established. Compared with the Mod group, the levels of TNF-α, IL-1β and IL-6 in the Car group, EGT group and Mix group were significantly reduced, indicating that L-carnosine, ergothioneine and the combination of the two can significantly reduce lung inflammation in COPD rats. At the same time, the Mix group was significantly better than the Car group and EGT group in reducing inflammatory factors TNF-α, IL-1β and IL-6, indicating that L-carnosine and ergothioneine synergistically enhance their efficacy and significantly reduce lung inflammation in COPD rats.

[0032] Figure 2 In the experiment, compared with the Con group, the expression of OCTN1 in the lung tissue of the Mod group was significantly reduced (p < 0.01). On the contrary, compared with the Mod group, the expression of OCTN1 in the Car group, the EGT group, and the Mix group was significantly increased, indicating that L-carnosine, ergothioneine, and the combination of the two can significantly increase the expression of OCTN1 in the lungs of COPD rats. At the same time, the effect of the Mix group on increasing OCTN1 was significantly better than that of the Car group and the EGT group, indicating that L-carnosine and ergothioneine synergistically enhance the efficacy and significantly increase the content of OCTN1 in the lungs of COPD rats.

[0033] Figure 3 In the experiment, compared with the Con group, the apoptosis rate of lung epithelial cells in the Mod group was significantly increased (p < 0.01). On the contrary, compared with the Mod group, the apoptosis rate of lung epithelial cells in the Car group, EGT group, and Mix group was significantly decreased, indicating that L-carnosine, ergothioneine, and the combination of the two can inhibit the apoptosis of lung epithelial cells in COPD rats. At the same time, the effect of Mix group in inhibiting apoptosis of lung epithelial cells was significantly better than that of Car group and EGT group, indicating that L-carnosine and ergothioneine synergistically enhance the efficacy and significantly inhibit the apoptosis of lung epithelial cells in COPD rats.

[0034] Mitochondria are the main production sites of endogenous reactive oxygen species, which can induce inflammatory responses. Mitochondria are also the central organelles in the endogenous caspase-induced apoptosis pathway. OCTN-1 is the transporter for ergothioneine to enter cells and mitochondria. After lung tissue is damaged, ergothioneine enters mitochondria through OCTN-1 in lung cells, directly scavenges free radicals, protects mitochondria, reduces inflammatory responses induced by mitochondrial dysfunction, and inhibits mitochondria-mediated apoptosis of lung epithelial cells. Compared with the Mod group, the expression of transporter OCTN-1 in the EGT group and the Mix group was significantly increased, indicating that more ergothioneine can enter cells and mitochondria through the transporter OCTN-1, directly scavenges reactive oxygen free radicals, plays an antioxidant role, and protects mitochondrial DNA, thereby reducing inflammation and apoptosis of lung epithelial cells. The Car group reduced the inflammation of lung tissue, and to a certain extent, also reduced the apoptosis of lung epithelial cells caused by inflammation. At the same time, the inflammatory factors and apoptosis rate of lung epithelial cells in the bronchoalveolar lavage fluid of the Mix group were significantly lower than those in the Car group and the EGT group, indicating that ergothioneine and L-carnosine can synergistically enhance their effectiveness and reduce the inflammatory factors and apoptosis rate of lung epithelial cells in the bronchoalveolar lavage fluid of COPD model rats, and the effect is better than that of a single component.

[0035] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. Use of a composition for improving lung diseases in the preparation of a drug for improving lung diseases, characterized in that, the composition for improving lung diseases comprises L-carnosine and ergothioneine in a mass ratio of (1~2):(1~3), and the lung diseases are chronic bronchitis or emphysema.

2. The use according to claim 1, characterized in that, the mass ratio of L-carnosine to ergothioneine is 1:

2.

3. The use according to claim 1, characterized in that, the dosage form of the drug is powder, tablet, pill, capsule, oral liquid, spray, powder inhaler, aerosol or nasal drops.

Images